Project Summary Rift Valley fever caused by Rift Valley fever virus (RVFV) is an acute, mosquito-borne, fever-causing zoonotic disease that affects both humans and animals. Large Rift Valley fever outbreaks have occurred throughout Africa and more recently in the Arabian Peninsula. Because RVFV is no longer restricted to African countries, it has raised concerns that the disease could spread worldwide. The RVFV is a select agent that requires high biocontainment facilities. This limitation has hampered the development of RVFV antivirals and vaccines. Despite the significant impact of the disease to the economy and public health, there are no fully licensed vaccine and antivirals available in the US for human and animal use. It is urgent to identify and develop effective inhibitors against RVFV to treat exposed and infected humans and animals. Dr. Ma?s group has developed a cell-based screening assay based on the RVFV MP12 vaccine strain that expresses Renilla Luciferase using Renilla as readout to identify RVFV inhibitors, and established a STAT1-KO mouse model susceptible to infection with MP12 vaccine strain that can be used in a BSL-2 facility. Furthermore, they have screened 727 compounds from the NIH collections of which two candidates including 6-azauridine and mitoxantrone inhibited replication of MP12. They hypothesize that effective inhibitors against RVFV can be identified by screening large compound collections and by further optimization of their structures and activities, and the mechanisms of inhibitory effects of identified candidates can be determined. Thus, they plan to use the developed high-throughput assay to identify inhibitors against RVFV, evaluate their efficacy in vitro and in mice, and understand the underlying mechanisms of inhibitory effects of identified candidates through two specific aims in this R21 proposal. In specific aim 1, the libraries assembled by the University of Kansas High Throughput Screening Laboratory that contain approximately 26,000 chemical compounds will be screened. The 26,000 compounds are predicted to cross the blood-brain barrier. Hits from the library will help overcome the challenge for RVFV antivirals to reach the brain through the blood-brain barrier to protect encephalitis. Primary hits will be confirmed by in vitro and in vivo assays. In specific aim 2, 6-azauridine and mitoxantrone as well as others identified in Aim 1 will be evaluated in vitro and in the BALB/c mice using virulent RVFV. To understand the underlying mechanisms of their inhibitory effects, whether the compounds block virus entry, inhibit virus replication and polymerase activity will be investigated using different designed assays. The results of this study could identify novel effective inhibitors against RVFV and understand the underlying mechanisms of their inhibitory effects, and offer novel insights toward the design of novel antiviral drugs against this zoonotic disease that will benefit both human and animal health.